Rhizocticins are phosphonate-containing oligopeptide antibiotics produced by the Gram-positive bacterium B. subtilis ATCC6633. They were originally discovered in 1949 based on their antifungal activity and collectively termed “rhizoctonia factor” (Michener and Snell, Arch. Biochem. 22, 208-214, 1949). The structures of rhizocticins were determined 40 years later (Rapp et al., Liebigs Ann. Chem., 655-661, 1988). They are dipeptide and tripeptide antibiotics consisting of a variable amino acid at the N-terminus followed by arginine and the non-proteinogenic amino acid (Z)-L-2-amino-5-phosphono-3-pentenoic acid (“APPA”, FIG. 1A). Interestingly, APPA is also the C-terminal amino acid of the tripeptide antibiotics plumbemycin A and B produced by Streptomyces plumbeus (FIG. 1B) (Park et al., Agric. Biol. Chem. 41, 573-579, 1977; Park et al., Agric. Biol. Chem. 41, 161-167, 1977). Plumbemycin A and B are tripeptides consisting of N-terminal alanine followed by aspartate (A) or asparagine (B) and the C-terminal non-proteinogenic amino acid APPA.
Rhizocticins enter the target fungal cell through the oligopeptide transport system. They are then cleaved by host peptidases to release APPA, which inhibits threonine synthase, an enzyme catalyzing the pyridoxal 5′-phosphate (PLP)-dependent conversion of phosphohomoserine to L-threonine (FIG. 1C). Hence, APPA interferes with the biosynthesis of threonine and related metabolic pathways, ultimately affecting protein synthesis and leading to growth inhibition. The inhibitory activity of APPA is due to the structural resemblance to phosphohomoserine, but possessing a hydrolytically stable C—P bond in place of the C—O—P moiety of phosphohomoserine.
Whereas rhizocticins exhibit antifungal activity, plumbemycins are antibacterials. It has been demonstrated that plumbemycins enter Escherichia coli K-12 via the oligopeptide transport system (Diddens, et al., J. Antibiot. 32, 87-90, 1979). As in the case of rhizocticins, L-threonine reverses the growth inhibition by plumbemycins in a concentration-dependent manner. Furthermore, similarly to rhizocticins, plumbemycins must be cleaved by peptidases of the target cell to release the active substance, APPA. The selectivity of these tripeptide antibiotics is thus not due to a difference in mode of action, but rather determined by the recognition of proteinogenic amino acids attached at the N-terminus of APPA by a specific oligopeptide transport system and/or peptidase. Furthermore, the target of APPA, threonine synthase, is not present in mammals, reducing the likelihood of toxicity to humans.
Due to the prevalence of infectious agents, and their effect on humans, there exists a need for anti-fungal and anti-bacterial agents, such as rhizocticin and plumbemycin. Unfortunately, the synthesis of APPA, as well as APPA-containing peptides, is a very challenging endeavor which makes it commercially impractical. Because of this limitation, and the inability to produce modified APPA-containing peptides from their native bacteria, these peptides are not presently a viable commercial option.